组合策略提高疏棉状嗜热丝孢菌脂肪酶在毕赤酵母中的分泌表达及其催化制备生物柴油的应用

[Objective]The aim of this study was to construction a recombinant Pichia pastoris to high level secretion expression Thermomyces lanuginosus lipase to reduce the cost of biocatalyst.In order to identify the catalytical activity,the obtained TLL was applied to produce biodiesel using Jatropha curcas oil as substrate.[Method]In this study,multiple strategies were used to improve the expression level of TLL in P.pastoris.Firstly,the effects of gene expression elements,including signal peptides,promoters and the gene doses,on lipase secretion expression were compared.Then,the fermentation parameters of the recombinant strain were regulated,including the initial pH of the culture medium,the induction temperature,the induction OD600,the methanol concentration and the addition of surfactant.To reduce the fermentation cost,crude glycerol,a by-product of biodiesel production,was used as the carbon source.In addition,in order to alleviate the inhibitory effect of methanol on cell growth,an additional expression cassette controlled by the constitutive promoter PGCW14 was introduced into the recombinant strain,and a one-step induction strategy was adopted.Because P.pastoris secreted less background protein,the obtained fermentation supernatant could be directly used as catalyst in biodiesel production.[Result]A recombinant P.pastoris with MFΔ signal peptide,4 copies of tll gene and co-expression of Vitreoscilla hemoglobin was constructed by genetic engineering.After 96 h of induction,1530 U/mL of lipase activity was obtained.The fermentation parameters were further optimized.Under the optimal fermentation parameters,the culture medium contains crude glycerol as the carbon source and 0.1%(w/v)Tween 80,the initial pH was 6.0,1%(v/v)methanol was added every 24 h for induction at 25℃when the OD600 was 3.0.After 192 h of fermentation,the total protein content in the fermentation supernatant was 1.2 g/mL,and the lipase activity reached 7700 U/mL.The effects on TLL expression were compared when methanol,methyl oleate and biodiesel were used as inducers.The highest expression level was observed when 1%(v/v)biodiesel was used as inducer.After fermentation for 192 h,the total protein content was 1.4 g/mL,and the lipase activity reached 8700 U/mL,which was 29-folid higher than that of the original strain.Moreover,the fermentation supernatant can be directly used in solvent-free system to produce biodiesel from J.curcas oil.The highest biodiesel yield of 96%was achieved after 60 h at 35℃with TLL concentration of 30 U/g oil,water content of 20%(w/w)and step-wise addition of methanol(methanol to oil ratio of 4:1).[Conclusion]When using crude glycerol as carbon source,TLL was efficiently expressed in P.pastoris by genetic engineering and fermentation regulation.The proposed one-step induction strategy could alleviate the growth inhibition caused by induction with high concentration of methanol.The fermentation supernatant could be directly used as biocatalyst.This study provides an efficient and low-cost biocatalyst for the biodiesel production by enzymatic route.